SLAM-seq reveals early transcriptomic response mechanisms upon glutamine deprivation in Chinese hamster ovary cells.

Mammalian cells frequently encounter subtle perturbations during recombinant protein production. Identifying the genetic factors that govern the cellular stress response can facilitate targeted genetic engineering to obtain production cell lines that demonstrate a higher stress tolerance. To simulate nutrient stress, Chinese hamster ovary (CHO) cells were transferred into a glutamine(Q)-free medium and transcriptional dynamics using thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) along with standard RNA-seq of stressed and unstressed cells were investigated. The SLAM-seq method allows differentiation between actively transcribed, nascent mRNA, and total (previously present) mRNA in the sample, adding an additional, time-resolved layer to classic RNA-sequencing. The cells tackle amino acid (AA) limitation by inducing the integrated stress response (ISR) signaling pathway, reflected in Atf4 overexpression in the early hours post Q deprivation, leading to subsequent activation of its targets, Asns, Atf3, Ddit3, Eif4ebp1, Gpt2, Herpud1, Slc7a1, Slc7a11, Slc38a2, Trib3, and Vegfa. The GCN2-eIF2α-ATF4 pathway is confirmed by a significant halt in transcription of translation-related genes at 24 h post Q deprivation. The downregulation of lipid synthesis indicates the inhibition of the mTOR pathway, further confirmed by overexpression of Sesn2. Furthermore, SLAM-seq detects short-lived transcription factors, such as Egr1, that would have been missed in standard experimental designs with RNA-seq. Our results describe the successful establishment of SLAM-seq in CHO cells and therefore facilitate its future use in other scenarios where dynamic transcriptome profiling in CHO cells is essential.

Manipulating gene expression levels in mammalian cell factories: An outline of synthetic molecular toolboxes to achieve multiplexed control.

Mammalian cells have developed dedicated molecular mechanisms to tightly control expression levels of their genes where the specific transcriptomic signature across all genes eventually determines the cell's phenotype. Modulating cellular phenotypes is of major interest to study their role in disease or to reprogram cells for the manufacturing of recombinant products, such as biopharmaceuticals. Cells of mammalian origin, for example Chinese hamster ovary (CHO) and Human embryonic kidney 293 (HEK293) cells, are most commonly employed to produce therapeutic proteins. Early genetic engineering approaches to alter their phenotype have often been attempted by "uncontrolled" overexpression or knock-down/-out of specific genetic factors. Many studies in the past years, however, highlight that rationally regulating and fine-tuning the strength of overexpression or knock-down to an optimum level, can adjust phenotypic traits with much more precision than such "uncontrolled" approaches. To this end, synthetic biology tools have been generated that enable (fine-)tunable and/or inducible control of gene expression. In this review, we discuss various molecular tools used in mammalian cell lines and group them by their mode of action: transcriptional, post-transcriptional, translational and post-translational regulation. We discuss the advantages and disadvantages of using these tools for each cell regulatory layer and with respect to cell line engineering approaches. This review highlights the plethora of synthetic toolboxes that could be employed, alone or in combination, to optimize cellular systems and eventually gain enhanced control over the cellular phenotype to equip mammalian cell factories with the tools required for efficient production of emerging, more difficult-to-express biologics formats.

In silico design of CMV promoter binding oligonucleotides and their impact on inhibition of gene expression in Chinese hamster ovary cells.

Modulation of expression levels of endogenous or recombinant genes can be of great interest for diverse applications, such as the study of genotype-phenotype relationships for a gene of interest, or fine-tuning of transcription to determine physiologically relevant effects of gene expression levels. During the last decades, several synthetic biology tools were established to control gene expression in mammalian cells such as Chinese hamster ovary (CHO) cells, one of the most important cell systems for basic research as well as the production of biopharmaceuticals. Here we describe the use of triplex forming oligos (TFOs), short RNA or ssDNA molecules that can bind to the major grove of their target duplex with great specificity, to control transgene expression in CHO cells. For proof of concept, a panel of TFOs with a size of 10-20 nts were designed with the help of the on-line tool Triplexator targeting the viral cytomegalovirus (CMV) promoter/enhancer region controlling the downstream reporter gene hCD4. The effect of TFOs was tested as ssDNA oligos pre-annealed to the promoter/enhancer region in vitro as well as upon endogenous transcription of the TFO as an RNA molecule binding to their target duplex in vivo. Results showed that not only binding of the TFO, but the exact location of triplex formation within the promoter/enhancer is paramount for transcription inhibition. After relieving a binding conflict by introducing a point mutation within the CMV promoter, longer TFOs (26-30 nts) could be designed and analysed. Selected TFOs achieved a reduction in recombinant hCD4 expression of up to 85% in CHO-K1 cells.